Severe hemolytic exacerbations of Chinese PNH patients infected SARS‐CoV‐2 Omicron

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolytic anemia, bone marrow failure, thrombophilia. COVID‐19, caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) with many variants including Omicron. Methods This study collected demographic and clinical data of 20 PNH patients with SARS‐CoV‐2 Omicron infection. Results They all were with high disease activity, and LDH level exceeded any documented since the diagnosis of PNH, and those reported in the literature for previously stable treatment with complement inhibitors. D‐dimer level elevated in 10 patients. 2 patients developed mild pulmonary artery hypertension. Glomerular filtration rate declined in 5 patients. 1 patient developed acute renal failure and underwent hemodialysis. Anemia and hemolysis were improved in 5 patients treated with eculizumab. Conclusions Hemolytic exacerbation of PNH with COVID‐19 is severe and eculizumab may be an effective treatment.


| INTRODUCTION
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired and clonal disease characterized by hemolytic anemia, bone marrow failure, thrombophilia, and multiorgan damage, which results from the mutation of the Xlinked PIGA gene. The blockade of glycosylphosphatidylinositol (GPI) synthesis caused by mutation results in the absence of GPI-anchored protein (such as CD55 and CD59), and CD55-deficient and CD59-deficient blood cells are more susceptible to complement attack and lysis. 1,2 Coronavirus disease 2019 (COVID-2019) first emerged in Wuhan, Hubei, China, by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3 SARS-CoV-2 was reported multiple variants, including Alpha, Beta, Gamma, Delta, Omicron, and Omicron shows a 13-fold increase in viral infectivity than Delta variant. 4 SARS-CoV-2 activate the complement system through the classical pathway, lectin pathway, and the alternative pathway. Specific antibody directed against the receptor-binding domain of the spike protein initiates the classical pathway, the binding of mannose-binding lectin with SARS-CoV-2 spike protein triggers the lectin pathway and SARS-CoV-2 spike protein may dysregulate the alternative pathway by binding heparan sulfate and competing with factor H, which is a negative regulator of complement activity. 5,6 After SARS-CoV-2 infection, patients with PNH more likely suffered the hemolysis, 7-16 patients usually presented visible hemoglobinuria and a small number patients showed pancytopenia. 17 Terminal complement inhibitors (such as eculizumab) are promising in COVID-19 treatment by blocking the formation of membrane attack complex and reducing proinflammatory and prothrombotic influence. 5,6,18 Due to the rarity of PNH, the clinical features of patients infected with SARS-CoV-2 were mostly case reports. China has been heavily affected by the SARS-CoV-2 Omicron outbreak with the peak in mid-December 2022. We collected clinical data from 20 PNH patients infected with SARS-CoV-2 Omicron and treated 5 patients with eculizumab.

| Diagnostic criteria
PNH patients were confirmed by flow cytometry using a reagent called fluorescent aerolysin (FLAER), which is the principal virulence factor of the bacterium Aeromonas hydrophilia, binds selectively and with high affinity to the GPI anchor. 2 Patients infected with COVID-19 were considered to have fever, cough, nasal congestion, sore throat, or other uncomfortable symptom and their reverse transcription polymerase chain reaction or antigen testing (by colloidal gold method) for SARS-CoV-2 was positive. 3 High disease activity (HDA) was defined as evidence of hemolysis (elevated lactate dehydrogenase [LDH] ≥1.5 times upper limit of normal [ULN]) and a history of at least one of the following signs or symptoms-fatigue, hemoglobinuria, abdominal pain, dyspnea, anemia (hemoglobin <100 g/L), MAVEs (including TEs), dysphagia, or erectile dysfunction. 19

| Methods
We collected the patient demographics, clinical manifestations, laboratory data 2,3 for understanding degree of anemia, hemolysis, thrombosis, organ damage, (including complete blood count [CBC], LDH, total bilirubin [TBIL], indirect bilirubin [IBIL], creatinine, glomerular filtration rate [GFR], blood urea nitrogen [BUN], D-dimer, IL-6, and pulmonary artery systolic pressure), treatment, and outcomes in the cohort. Data on 20 patients with PNH registered in four centers is to evaluate the occurrence and clinical characteristics of SARS-CoV-2 infection, and the efficacy of eculizumab.

| Laboratory tests
All laboratory tests were done by the medical centers where the individual patients visited.
CBC used EDTA as anticoagulants by blood cell analyzer following the two principles of electricity (electrical impedance method and radio frequency conductance method) and optical (laser scattering method and spectrophotometry). LDH, TBIL, IBIL, creatinine, GFR, and BUN used heparin as anticoagulants by a biochemical analyzer following the principle of photoelectric colorimetry. D-dimer used sodium citrate as anticoagulants detected by the technique of immunofluorescence.
IL-6 used EDTA as anticoagulants detected by the technique of chemiluminescence.

| Statistics analysis
Categorical variables were described using frequencies and percentages, and continuous variables were described using median, minimum, and maximum. Shapiro-Wilk test is used to test whether the data conform to a normal distribution.  Mild pulmonary hypertension was found in 2 patients (32 and 35 mmHg, respectively). The GFR in 5 patients (25%, 5/20) was lower than 60 mL/min/1.73 m. 2 One patient underwent hemodialysis due to acute renal failure.

| Outcomes of COVID-19 and PNH
In our study, 12 of 20 (60%) were hospitalized. Two (2/20, 10%) patients developed severe COVID-19-one with severe viral pneumonia but recovered after two doses of eculizumab with oxygen support and the other with acute renal failure but recovered after hemodialysis.
Of the 5 patients using eculizumab, 2 were not in HDA status. But 13 of the other 15 patients remained in HDA status during the second week after infection with SARS-CoV-2 Omicron.

| DISCUSSION
PNH is a complement-mediated hemolytic anemia 1 and SARS-CoV-2 activates complement and inflammatory factor storm. 6 PNH patients infected with SARS-CoV-2 are more likely to have hemolytic episodes. Twenty patients all had HDA in this study and had severe hemolysis and anemia: LDH, IBIL increased and red blood cell count, hemoglobin declined significantly.
Current study found that Omicron-induced hemolysis was more intense than ever happened since diagnosis. On the one hand, SARS-CoV-2 triggered complement activation through three pathways 5,6 and on the other hand, coronavirus infection activates monocyte, macrophage, and dendritic cell, then releases IL-6 and amplifies cytokine cascade. 20 Under dual attack, it causes severe hemolysis in patients suffering from PNH. In this study, there was no thromboembolism, but D-dimer values of a half of patients were beyond the ULN. Zlatko et al. 16 reported 1 PNH patient who presented with deep vein thrombosis as the first sign of COVID-19. SARS-CoV-2 may increase thrombophilia in PNH in the context of multiple triggers, such as increased inflammatory factors, endothelial injury, platelet, and thrombin activations. Acute kidney injury of PNH will be caused by a variety of reasons, such as the direct toxicity of free hemoglobin released by broken red blood cells, the constriction of renal blood vessels caused by NO consumption, and the direct damage caused by the coronavirus and cytokine storm. 2,21,22 In our cohort, there were 5 patients with severe renal function decline (GFR ≤60 mL/min/1.73 m 2 ), even 1 accepted hemodialysis.
C5 inhibitors have been recommended as the first line treatment for PNH, 23 greatly improving the poor prognosis of PNH. C5 inhibitors protect PNH clone from attack by blocking the complement activation pathway and significantly improve hemolytic anemia, reduce events of thrombosis, and alleviate damage of renal function. [24][25][26][27] Meanwhile, eculizumab has been used to treat severe COVID-19. 28 Excess C5a induces the release of proinflammatory cytokines from innate immune cells which is thought to play a key role in acute lung injury. 29 After eculizumab stops the cleavage of C5, the production of C5a is reduced. In an Annane's study 30 of 80 patients with severe COVID-19, 35 patients treated with additional eculizumab showed higher survival rate (82.9% vs. 62.2%, p = .04) and improved tissue oxygenation compared with 45 patients who received supportive care alone. Another case reported that after Diurno 31 administrated eculizumab to 4 patients with severe COVID-19, their inflammatory markers declined and recovery time was cut short.
The literature 10,[12][13][14]16 documented less frequent hemolysis following SARS-CoV-2 infection in PNH patients who regularly used complement inhibitors. Compared with cases with LDH values reported in the literature, hemolysis in current study was more severe (LDH 7.47 × ULN vs. 2.04 × ULN, p < .001). In our study, 5 patients receiving eculizumab achieved improvement in hemolysis and anemia, and COVID-19 was also relieved. Eculizumab maybe a good choice for patients with PNH after SARS-CoV-2 infection.
In conclusion, our study preliminarily demonstrate SARS-CoV-2 infection could induce a hemolytic exacerbation in patients with PNH but eculizumab can effectively control acute hemolysis. It is the unavoidable association between PNH hemolytic exacerbation and visiting hospital. A large amount of long-term follow-up data are still needed to assess the impact of SARS-CoV-2 on patients with PNH, evaluate the dose and duration of eculizumab, and develop better prevention and control plans.